Anthrax is a zoonotic illness that has been recognized for centuries. In the 1870s, Robert Koch demonstrated for the first time the bacterial origin of a specific disease, with his studies on experimental anthrax, and also discovered the spore stage that allows persistence of the organism in the environment. Shortly afterward, Bacillus anthracis was recognized as the cause of woolsorter disease, now known as inhalational anthrax. The development of vaccines against anthrax began in 1880 with William Greenfield's successful immunization of livestock against anthrax and Louis Pasteur's 1881 trial of a heat-cured anthrax vaccine in sheep.
Bacillus anthracis is a large, gram-positive, sporulating rod, with square or concave ends. Growing readily on sheep blood agar, B. anthracis forms rough, gray-white colonies of four to five micrometer, with characteristic comma-shaped or “comet-tail” protrusions. Several tests are helpful in differentiating B. anthracis from other Bacillus species. Bacillus anthracis is characterized by an absence of the following: hemolysis, motility, growth on phenylethyl alcohol blood agar, gelatin hydrolysis, and salicin fermentation. Bacillus anthracis may also be identified by the API-20E and API-50CHB systems used in conjunction with the previously mentioned biochemical tests. Definitive identification is based on immunological demonstration of the production of protein toxin components and the poly-D-glutamic acid capsule, susceptibility to a specific bacteriophage, and virulence for mice and guinea pigs.
Naturally occurring human cases of anthrax are invariably zoonotic in origin, with no convincing data to suggest that human-to-human transmission has ever taken place. Primary disease takes one of three forms: (1) cutaneous, the most common, which results from contact with an infected animal or animal product; (2) inhalational, which is much less common and results from spore deposition in the lungs; and (3) gastrointestinal, which is due to ingestion of infected meat. Most literature cites cutaneous disease as constituting the large majority (up to 95%) of naturally occurring exposure cases.
Anthrax has been studied for use as a biological weapon for over 80 years, with the transmission of spores through air as the most likely method of transmission, resulting in inhalational anthrax. Due to the rapidly fatal hemorrhagic mediastinitis caused by inhalation of anthrax spores, the dissemination of airborne spores in a populated area could be devastating.
Disease occurs when spores enter the body, germinate to the bacillary form, and multiply. In cutaneous disease, spores gain entry through cuts, abrasions, or in some cases through certain species of biting flies. Germination is thought to take place in macrophages, and toxin release results in edema and tissue necrosis but little or no purulence, probably because of inhibitory effects of the toxins on leukocytes. Generally, cutaneous disease remains localized, although if untreated it may become systemic in up to 20% of cases, with dissemination via the lymphatics. In the gastrointestinal form, B. anthracis is ingested in spore-contaminated meat, and may invade anywhere in the gastrointestinal tract. Transport to mesenteric or other regional lymph nodes and replication occur, resulting in dissemination, bacteremia, and a high mortality rate. As in other forms of anthrax, involved nodes show an impressive degree of hemorrhage and necrosis.
The pathogenesis of inhalational anthrax is more fully studied and understood than that of cutaneous or gastrointestinal anthrax. Inhaled spores are ingested by pulmonary macrophages and carried to hilar and mediastinal lymph nodes, where they germinate and multiply, elaborating toxins and overwhelming the clearance ability of the regional nodes. Bacteremia occurs, and death soon follows.
Penicillin remains the drug of choice for treatment of susceptible strains of anthrax, with ciprofloxacin and doxycycline employed as suitable alternatives. Some data in experimental models of infection suggest that the addition of streptomycin to penicillin may also be helpful. Penicillin resistance remains extremely rare in naturally occurring strains; however, the possibility of resistance should be suspected in a biological warfare attack. Since reports in 1999 that an anthrax strain had been engineered to be resistant to the tetracycline and penicillin classes of antibiotics, ciprofloxacin is the recommended treatment for adults with suspected inhalational anthrax. The more severe forms require intensive supportive care and have a high mortality rate despite optimal therapy.
The virulence of B anthracis is mediated by two plasmids, pXO1 and pXO2, which encodes genese involved in toxin production and capsule formation, respectively. The pXO1 genes pagA, lef, and cya encode the tripartite toxin protective antigen (PA)-lethal factor (LF)-edema factor (EF) associated with B. anthracis pathogenicity (Inglesby 2002). Production of PA-LF-EF peaks during the late exponential phase of vegetative growth (Liu 2004). The importance of a toxin in pathogenesis was demonstrated in the early 1950s, when sterile plasma from anthrax-infected guinea pigs caused disease when injected into other animals (Smith and Keppie 1954). It has since been shown that the anthrax toxins are composed of three entities, which in concert lead to some of the clinical effects of anthrax (Stanley and Smith 1961; Beall 1962). The first of these, protective antigen (PA), is an 83 kd protein so named because it is the main protective constituent of anthrax vaccines. Protective antigen binds to a cellular receptor (Bradley 2001) and is proteolytically cleaved by cell surface furin to produce a 63-KD fragment (PA63). A second binding domain is then exposed on the 63 kd remnant, which combines with either edema factor (EF), an 89 kd protein, to form edema toxin, or lethal factor (LF), a 90 kd protein, to form lethal toxin (Leppla 1990). This occurs through the receptor-bound PA63, which oligomerizes to a heptamer and acts to translocate the catalytic moieties of the toxin, LF and/or EF, from endosomes to the cytosol (Singh 1999). Edema factor, a calmodulin-dependent adenylate cyclase, acts by converting adenosine triphosphate to cyclic adenosine monophosphate. Intracellular cyclic adenosine monophosphate levels are thereby increased and neutrophil functions are impaired, leading to the edema characteristic of the disease (Leppla 1982; Swartz 2001; Inglesby 2002). Lethal factor appears to be a zinc metalloprotease and has been demonstrated to lyse macrophages at high concentration (Bradley 2001), while inducing the release of tumor necrosis factor .alpha. and interleukin 1.beta. at lower concentrations (Friedlander 1986; Hanna 1993), which have been linked to the sudden death in anthrax infection (Swartz 2001; Inglesby 2002).
Although anthrax vaccination dates to the early studies of Greenfield and Pasteur, the “modern” era of anthrax vaccine development began with a toxin-producing, unencapsulated (attenuated) strain in the 1930s. Administered to livestock as a single dose with a yearly booster, the vaccine was highly immunogenic and well tolerated in most species, although somewhat virulent in goats and llamas. This preparation is essentially the same as that administered to livestock around the world today. The first human vaccine was developed in the 1940s from nonencapsulated strains. This live spore vaccine, similar to Sterne's product, is administered by scarification with a yearly booster. Studies show a reduced risk of 5- to 15-fold in occupationally exposed workers (Shlyakhov and Rubinstein 1994).
To date, there have been many attempts to improve the safety profile and immunogenicity of anthrax vaccines by using PA as an antigen. These attempts include the formulation of PA in adjuvants (Ivins 1992), the use of purified PA (Singh 1998), the development of PA-based DNA vaccines (Gu 1999), and the expression of PA in Salmonella typhimurium (Coulson 1994).
British and U.S. vaccines were developed in the 1950s and early 1960s, with the U.S. producing an aluminum hydroxide-adsorbed, cell-free culture filtrate of an unencapsulated strain (V770-NP 1-R) containing PA as the major protective immunogen, and the British vaccine an alum-precipitated, cell-free filtrate of a Sterne strain culture. The U.S. vaccine has been shown to induce high levels of antibody only to protective antigen, while the British vaccine induces lower levels of antibody to protective antigen but measurable antibodies against lethal factor and edema factor (Turnbull 1986; Turnbull 1988). Neither vaccine has been examined in a human clinical efficacy trial (Inglesby 2002). A high number of the recipients of the vaccine have reported some type of reaction to vaccination although most were minor. Manufacturer labeling for the current Michigan Department of Public Health anthrax vaccine adsorbed (AVA) product cites a 30% rate of mild local reactions and a 4% rate of moderate local reactions with a second dose. The current complex dosing schedule for the AVA vaccine consists of 0.5 mL administered subcutaneously at 0, 2, and 4 weeks, and 6, 12, and 18 months, followed by yearly boosters. Animal studies examining the efficacy of available anthrax vaccines against aerosolized exposure have been performed. While some guinea pig studies question vaccine efficacy, primate studies have supported its role. In recent work, rhesus monkeys immunized with 2 doses of the AVA vaccine were challenged with lethal doses of aerosolized B anthracis spores. All monkeys in the control group died 3 to 5 days after exposure, while the vaccinated monkeys were protected up to 2 years after immunization (Ivins 1996). Another trial used the AVA vaccine in a 2-dose series with a slightly different dosing interval, and again found it to be protective in all rhesus monkeys exposed to lethal aerosol challenge (Pitt 1996). Thus, available evidence suggests that two doses of the current AVA vaccine should be efficacious against an aerosol exposure to anthrax spores. However, one significant limitation on the use of vaccines is that existing vaccines provide no protection against a number of strains of B. anthracis. Additionally, the current requirement for multiple injections resulting local pain and edema suggests an effective alternative is needed (Joellenbeck 2002). The Georgian/Russian anthrax vaccine consists of live spores from a Sterne strain of B. anthracis that is administered in the shoulder by scarification. This vaccine also has several undesirable side effects and unknown efficacy (Demicheli 1998). Other alternatives that have been investigated include a highly purified, minimally reactogenic, recombinant protective antigen vaccine using aluminum as well as other adjuvants, cloning the protective antigen gene into a variety of bacteria and viruses, and the development of mutant, avirulent strains of B. anthracis. 
Further underscoring the need for a new class of anthrax vaccines or remedies is the development of enabling technology capable of engineering B. anthracis spores into vectors expressing unpredictable toxins by replacing pXO1 with an artificial plasmid, owing to the dispensability of the pXO1 plasmid for growth of B. anthracis (Welkos 2001).
In the 2001 U.S. anthrax attacks, anthrax spores were enclosed in letters and envelopes sent through the mail and resulted in both cutaneous anthrax (11 cases: 7 confirmed, 4 suspected) in those who handled such letters, and inhalational anthrax (11 cases) (Centers for Disease Control and Prevention 2001). Other known experiences with large-scale, non-naturally occurring anthrax exposure are limited to the 1979 accidental release of anthrax spores from a bioweapons factory in Sverdlovsk, Russia.
These recent incidents, which also include the suspected use of biological and chemical weapons during the Persian Gulf War, underscore the threat of biological warfare either on the battlefield or by terrorists. Anthrax has been the focus of much attention as a potential biological warfare agent for at least six decades, and modeling studies have shown the potential for use in an offensive capacity. Dispersal experiments with the simulant Bacillus globigii in the New York subway system in the 1960s suggested that release of a similar amount of B. anthracis during rush hour would result in 10,000 deaths. On a larger scale, the World Health Organization estimated that 50 kg of B. anthracis released upwind of a population center of 500,000 would result in up to 95,000 fatalities, with an additional 125,000 persons incapacitated (Huxsoll 1989). Both on the battlefield and in a terrorist strike, B. anthracis has the attribute of being potentially undetectable until large numbers of seriously ill individuals present with characteristic signs and symptoms of inhalational anthrax. Given these findings, efforts to prevent the disease or to ameliorate or treat its effects are of obvious importance. The U.S. military's current M17 and M40 gas masks provide excellent protection against the 1 to 5 .mu.m particulates needed for a successful aerosol attack. Assuming a correct fit, these masks would be highly effective if in use at the time of exposure. Some protection might also be afforded by various forms of shelter.
Until recently, the AVA anthrax vaccine was supplied only to the Department of Defense for vaccination of soldiers. This use has recently expanded to include vaccination of reporters who would face potential exposure while covering any future warfare or terrorist situations. At this time, vaccination of the general public is not encouraged by the Centers for Disease Control.
Due to the limited use of the available anthrax vaccines and their limited ability to prevent infection caused by numerous anthrax strains, it is therefore apparent that while certain prophylactic and treatment schemes may prove useful in preventing or ameliorating anthrax infections, there remains a compelling need to improve the arsenal of techniques and agents available for this purpose.
Contemporary anti-anthrax remedies focus on the three-component toxin system protective antigen (PA)-lethal factor (LF)-edema factor (EF) that is produced during multiplication of the vegetative form of B. anthracis in the host (Mock and Fouet 2001). The dissemination of an odorless and invisible aerosol containing PA-free anthrax spores encoding exogenous toxins would be devastating, as all PA-targeted anthrax vaccines (Price 2001; Welkos 2001; Joellenbeck 2002; Rhie 2003; Tan 2003) and remedies (Sellman 2001) are ineffective in protection against anthrax strains without PA. Furthermore, although targeting PA has proven effective to varying degrees of success in counteracting anthrax, it is unknown at this time whether any of the PA-targeted methods can protect humans against inhalational anthrax (Inglesby 2002) during a massive onslaught with airborne anthrax spores used as a bioweapon.
A solution to this dilemma might be found at the level of spore germination and early outgrowth.
The present invention identifies B. anthracis spore proteins as novel targets in decontamination, immunoprophylaxis, and post-exposure therapy against anthrax.
During the life cycle of B. anthracis, the germinating spores are likely the weakest link in the cycle, akin to plant seedlings and animal babies, and hence are the easiest vaccine target. Additionally, germination is an upstream event during the life cycle of B. anthracis; arrest of spore germination will preclude any downstream events including the production of PA-LF-EF. Consequently, blocking spore germination and early outgrowth provides a logical means to arrest bioengineered anthrax spores regardless of the exogenous toxin they may encode.
Additionally as previously mentioned, PA, LF, and EF are encoded by a plasmid in B. anthracis. Technology that enables the replacing of this plasmid with others encoding other unpredictable toxins (e.g., tetanus toxin, cobratoxin, etc.) is not beyond reach. Bioterrorists may bypass any PA-targeted remedies by launching an attack with anthrax spores producing toxins other than PA-LF-EF. Therapies that target the germinating spores would prevent the production of such toxins from occurring. Arrest of spore germination with vaccines and/or inhibitors overall may prevent utilization of anthrax spores as bioweapons.
Furthermore, factors associated with spore germination or outgrowth may be highly conserved and can hardly be altered, making these proteins an ideal target that should be conserved between strains. In contrast, vegetative cells undergo mutations over time. Development of antibiotic resistance in vegetative cells is one such example.
The present invention also provides a new composition for vaccination against Bacillus anthracis, using adenovirus and bacterium vectored nasal and epicutaneous vaccines that can provide protection to large numbers of people in a timely manner by non-medical personnel.
There are several noteworthy reasons for utilizing recombinant Ad vector as a vaccine carrier. These include (i) Ad vectors are capable of transducing both mitotic and postmitotic cells in situ (Shi 1999), (ii) stocks containing high titers of virus (greater than 10.sup.11 pfu [plaque-forming units] per ml) can be prepared, making it possible to transduce cells in situ at high multiplicity of infection (MOI), (iii) the vector is safe based on its long-term use as a vaccine, (iv) the virus is capable of inducing high levels of transgene expression (at least as an initial burst), and (v) the vector can be engineered to a great extent with versatility. Recombinant Ad vectors have been utilized as vaccine carriers by intranasal, epicutaneous, intratracheal, intraperitoneal, intravenous, subcutaneous, and intramuscular routes.
Ad-vectored nasal vaccine appears to be more effective in eliciting an immune response than injection of DNA or topical application of Ad (Shi 2001). Previously reported results have shown that the potency of the E1/E3-defective Ad5 vector as a nasal vaccine carrier is not suppressed by any preexisting immunity to Ad (Xiang 1996; Shi 2001).
Furthermore, it is possible to create an epicutaneous vaccine using recombinant Escherichia coli vectors as the carrier. Expression of heterologous genes in recombinant E. coli vectors about two decades ago (Itakura 1977; Goeddel 1979; Goeddel 1979) allowed E. coli cells to be utilized as protein factories for production of exogenous proteins including a variety of vaccines. It was subsequently demonstrated that recombinant plasmid DNA extracted from E. coli vectors could be inoculated into animals to elicit an immune response against antigens encoded by the plasmid, the so called genetic immunization or DNA-based vaccination (Tang 1992; Ulmer 1993). Both approaches required the disruption of E. coli cells prior to inoculation into animals, in conjunction with subsequent extraction and purification of recombinant protein and DNA, respectively; it is hazardous to inject undisrupted E. coli cells into humans as a vaccine due to the presence of endotoxin. It has recently been demonstrated that topical application of live or irradiated E. coli cells may be a more potent vaccination modality than injection of DNA. It is also believed that the skin is able to disrupt E. coli cells following topical application and the present invention hypothesizes that the antigen may be captured from disrupted E. coli cells in the outer layer of skin followed by antigen presentation and the elicitation of protective immunity against pathogens, including Bacillus anthracis. Topical application of E. coli cells as a vaccination modality does not pose a biosafety concern because the skin is already in frequent contact with E. coli cells in the environment. Moreover, the biosafety margin of this modality can be further amplified by making recombinant E. coli vectors replication incompetent, for example, with .gamma.-irradiation.